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. 2022 Jun 8;11(6):1129.
doi: 10.3390/antiox11061129.

Bioenergetic and Autophagic Characterization of Skin Fibroblasts from C9orf72 Patients

Maria Isabel Alvarez-Mora  1   2   3 , Gloria Garrabou  2   4 , Tamara Barcos  1 , Francisco Garcia-Garcia  5 , Ruben Grillo-Risco  5 , Emma Peruga  1 , Laura Gort  1   2   3 , Sergi Borrego-Écija  6 , Raquel Sanchez-Valle  3   6 , Judith Canto-Santos  2   4 , Paula Navarro-Navarro  1 , Laia Rodriguez-Revenga  1   2   3
Affiliations

Bioenergetic and Autophagic Characterization of Skin Fibroblasts from C9orf72 Patients

Maria Isabel Alvarez-Mora et al. Antioxidants (Basel). .

Abstract

The objective of this study is to describe the alterations occurring during the neurodegenerative process in skin fibroblast cultures from C9orf72 patients. We characterized the oxidative stress, autophagy flux, small ubiquitin-related protein SUMO2/3 levels as well as the mitochondrial function in skin fibroblast cultures from C9orf72 patients. All metabolic and bioenergetic findings were further correlated with gene expression data obtained from RNA sequencing analysis. Fibroblasts from C9orf72 patients showed a 30% reduced expression of C9orf72, ~3-fold increased levels of oxidative stress and impaired mitochondrial function obtained by measuring the enzymatic activities of mitochondrial respiratory chain complexes, specifically of complex III activity. Furthermore, the results also reveal that C9orf72 patients showed an accumulation of p62 protein levels, suggesting the alteration of the autophagy process, and significantly higher protein levels of SUMO2/3 (p = 0.03). Our results provide new data reinforcing that C9orf72 cells suffer from elevated oxidative damage to biomolecules and organelles and from increased protein loads, leading to insufficient autophagy and an increase in SUMOylation processes.

Keywords: C9orf72; SUMOlyation; autophagy; bioenergetics.

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Conflict of interest statement

R.S.-V. has served at scientific advisory boards from Ionnis Pharmaceuticals and Wave Life Science. The other authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Analysis of C9orf72 protein levels in C9ALS/FTD patients (n = 4) and the control samples (n = 4) showed a 30% reduction in protein content in the patients. Results are expressed as means ± standard error of the mean (SEM).
Figure 2
Figure 2
Oxidative stress measured through (A) lipid and (B) protein peroxidation in C9ALS/FTD patients and the control samples. C9ALS/FTD patients exhibited an upward trend in lipid peroxidation compared to the control fibroblasts and a significantly increase in protein oxidation levels (oxidative index as protein carbonyls). The results are expressed as means ± standard error of the mean (SEM). * p < 0.05.
Figure 3
Figure 3
Autophagic flux in C9ALS/FTD patients and the control fibroblasts. (A) p62 and (B) LC3BII protein level at basal conditions (0 h) and under bafilomycin A1 treatment (4 or 8 h). The results are expressed as means ± standard error of the mean (SEM). No statistically significant results were obtained, although p62 protein levels were 2-fold increase in C9ALS/FTD patients compared to the control fibroblasts.
Figure 4
Figure 4
Mitochondrial bioenergetics in C9ALS/FTD patients and the control samples. VDAC protein content was quantified by Western blot and represented as a ratio of β-actin content. The results are expressed as means ± standard error of the mean (SEM). Although not statistically significant, fibroblasts from C9ALS/FTD patients showed a 2-fold increase in VDAC content at basal conditions and after bafilomycin treatment (4 h and 8 h).
Figure 5
Figure 5
Fibroblasts from C9ALS/FTD patients show increased SUMO2/3 protein content. The results are expressed as means ± standard error of the mean (SEM). Significant p-values were considered for p < 0.05 (*).
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